EAGER: Collaborative Research: Tracking of KOR1 Protein Transport in Arabidopsis using Fluorescent-Timer Imaging System

在这个项目中，将开发一种方法来直接观察蛋白质KORRIGAN1(KOR1)在植物细胞中的运动。KOR1在纤维素纤维的形成中起作用，而纤维素纤维是植物细胞壁的主要成分。由KOR1控制的纤维素纤维沉积影响细胞生长和生物量生产。KOR1在细胞内从一个隔室移动到另一个隔室，最后到达细胞表面，最终在那里沉积纤维素纤维。为了可视化KOR1在植物细胞中的动态，该项目将应用荧光计时器(FT)技术，在该技术中，每个KOR1蛋白都被标记上一个时间敏感的荧光标签。因为FT的颜色会随着时间的推移而改变，所以表达FT标记的KOR1的细胞的荧光图像将允许可视化KOR1在细胞内的年龄和位置。这将揭示KOR1通过植物细胞的运输途径。通过观察KOR1转运在不同生物环境下的变化，这些研究将揭示某些因素如何影响蛋白质在细胞内的运输路径。这是一种新的植物生物学研究工具/方法，可以改变科学家看待任何植物细胞中蛋白质的方式。在植物中，KOR1在不同的亚细胞域中的分布被严格保持，如跨高尔基网络(TGN)、质膜(PM)、液泡膜(TP)以及分裂细胞时的细胞板。KOR1的功能障碍通常与TP靶向性增加有关。该项目旨在将KOR1运输的动态可视化，而不仅仅是分析终点位置。将研究与KOR1生物学相关的两个运输过程。首先，通过追踪KOR1蛋白表达诱导后的年龄，分析从头合成的KOR1的运输途径/动力学和重定向机制。接下来，将利用KOR1的年龄信息来剖析KOR1在细胞分裂过程中到细胞板的多条靶向路线。这些系统将作为模型，进一步剖析从数量上影响KOR1运输的各种因素。跨学科团队将优化植物细胞生物学的FT技术作为一个整体的荧光分析包，包括具有植物成熟时间特征的FT、兼容的荧光亚细胞标记物和自动化图像处理管道。本项目建立的实验平台将作为进一步研究影响KOR1转运的多种因素的基础，包括蛋白质N-糖基化途径以及与纤维素合成酶复合体的相互作用。

In this project, methods will be developed to allow direct observation of the movement of a protein, KORRIGAN1 (KOR1), in plant cells. KOR1 functions in the formation of cellulose fibers, which are the primary constituents of plant cell walls. Cellulose fiber deposition that is controlled by KOR1 influences cell growth and biomass production. KOR1 travels from one compartment to another inside cells before it finally reaches the cell surface where cellulose fibers will ultimately be deposited. To visualize the dynamics of KOR1 in plant cells, this project will apply fluorescent-timer (FT) technology, in which each KOR1 protein is labeled with a time-sensitive fluorescent tag. Because FT changes its color over time, fluorescence images of cells expressing FT-tagged KOR1 will allow visualization of the age and location of KOR1 inside of the cells. This will reveal the route of KOR1 transport through the plant cell. By observing how KOR1 transport changes under different biological circumstances, these studies will reveal how certain factors influence the paths that proteins travel within cells. This is a novel research tool/approach in plant biology that can change the way the scientists view proteins in any plant cell.In plants, distribution of KOR1 among different subcellular domains, such as trans-Golgi network (TGN), plasmamembrane (PM), tonoplast (TP), as well as the cell plate in case of the dividing cells, is scrupulously maintained. Dysfunction of KOR1 is often associated with increased targeting to TP. This project aims to visualize the dynamics of KOR1 transport beyond analysis of endpoint locations. Two transport processes relevant to KOR1 biology will be studied. First, transport route/kinetics and a redirection mechanism of de-novo-synthesized KOR1 will be analyzed by tracking the age of KOR1 after induction of protein expression. Next, KOR1 age information will be used to dissect the multiple targeting routes of KOR1 to the cell plate during cell division. These systems will serve as models for further dissection of various factors that quantitatively affect KOR1 transport. The interdisciplinary team will optimize FT technology for plant cell biology as a whole fluorescence analysis package, including FT with characterized in planta maturation time, compatible fluorescent subcellular markers, and automated image-processing pipelines. The experimental platform established in this project will serve as the basis for further investigation of multiple factors that affect KOR1 transport, including protein N-glycosylation pathways and interactions with cellulose synthase complex.